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Research Article

Antioxidant and calcium levels in mature and immature diabetic

cataract lens
Deepa K 1, *, Manjunatha Goud B.K 2, Suma M.N. 1, Devaki R.N. 1 , Nandini M. 3 and Sudhir 4
1
Department of Biochemistry, JSS Medical College, JSS University, Mysore, India. 2 Department of Biochemistry, Ras AI Khaimah Medical
and Health Sciences University, UAE. 3 Department of Biochemistry, Kasturba Medical College, Mangalore, India. 4 Department of Community
Medicine, JSS Medical College, JSS University, Mysore, India.

Corresponding author ABSTRACT

Email: Cataract is considered a major cause of visual impairment in diabetic patients as the incidence and
drdeepakrishna@yahoo.co.in progression of cataract is elevated in patients with diabetes mellitus. The pathogenesis of diabetic
cataract development is still not fully understood. Due to increasing numbers of type 1 and type 2
diabetics worldwide, the incidence of diabetic cataracts steadily rises. Even though cataract
Phone No:
surgery, the most common ophthalmic procedure worldwide, is an effective cure, the elucidation of
+91 - 9900595772
patho- mechanisms to delay or prevent the development of cataract in diabetic patients remains a
challenge. Furthermore, patients with diabetes mellitus have higher complication rates from
Fax No: cataract surgery. Both diabetes and cataract pose an enormous health and economic burden,
-- particularly in developing countries, where diabetes treatment is insufficient and cataract surgery
often inaccessible. Hence the objective of the study was to assess the antioxidant levels and
Editor calcium levels in different stages of cataract. Our study showed a significant reduction in lens
Ganesh Kumar Agrawal antioxidant activity with significantly increased calcium levels in mature stage of cataract,
suggesting decreased antioxidant activity plays an important role in diabetic cataract by causing
DOI deposition of calcium in lens.
dx.doi.org/10.3126/ijls.v6
Key words: Diabetes; Cataract; Antioxidant; Calcium
i1.5290

INTRODUCTION Diabetic cataract is more prevalent in diabetic

Worldwide more than 285 million people are
affected by diabetes mellitus. This number is expected
to increase to 439 million by 2030 according to the
International Diabetes Federation.
Diabetes mellitus is characterized by absolute
or relative deficiencies in insulin secretion and/or
insulin action associated with chronic hyperglycemia
and disturbances of carbohydrate, lipid and protein
metabolism. During diabetes, persistent
hyperglycemia causes increased production of free
radicals especially reactive oxygen species (ROS), for
all tissues from glucose auto-oxidation and protein
glycosylation (Aragno et al., 1999). Free radicals are
generated as by-products of normal cellular
metabolism; however, several conditions are known
to disturb the balance between ROS production and
cellular defense mechanisms (Bonnefont et al., 2000).
adults compared to non diabetics. It depends on
duration of diabetes and glycemic status. It
progresses rapidly if the blood sugar levels are not
maintained within normal range. The mechanism
behind the diabetic cataract is mainly due to
hyperglycemia induced oxidative stress. Excessive
levels of glucose reaching the mitochondria leads to
an overdrive of electron transport chain, resulting in
over production of superoxide anions, normally
scavenged by mitochondrial superoxide dismutase.
When latter fails oxidative stress develops and it is
proposed that this mechanism is responsible for
activation of all major pathways responsible for
diabetic complications of which includes, activation of
sorbitol pathway, glycation and protein kinase
activation (Brownlee, 2000).
Hyperglycemia can also stimulate oxidative
stress by the auto oxidation of glucose in the presence

56 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
Research Article
of transition metals as well as generation of ROS
during the process of glycation. Oxidative stress also
affects the expression of antioxidant enzymes by its
action on signal transduction. Hyperglycemia can also
cause inactivation of the existing enzymes by
glycation. Thus oxidative stress may develop by
insufficient antioxidant activity even if ROS
production is within physiological range (King et al.,
1996)
Calcium is an important extracellular cation in
the body, total calcium in the body is about 1-1.5kg.
99% is in bone and 1% in extracellular fluid. It is
present both in extracellular and intracellular
compartments. The cell membrane is generally
impermeable to calcium ions. Normally the calcium
levels are maintained at low concentration inside the
cell. This is maintained by Na+ K+ exchanger; Na+ Ca2+
exchanger and calcium ATPase pump (Galvan, 1988).
Optimum intracellular calcium is necessary to
maintain lens transparency. Calcium is maintained in
micromoles range in the cytoplasm by regulatory
systems. In normal lens, the ionized calcium is less
than 1% of the total concentration and below 0.1% of
the level in surrounding aqueous humor (Tomlinson
et al., 1991).
Metal ions are known to play an essential role
in living systems, both in growth and in metabolism.
Impaired metabolism of trace elements is observed in
diabetic patients. Alteration in the capacity to
maintain normal calcium homeostasis have been
suggested to underlie the reduced cellular functions
characteristic of aging process and predisposes the
senescent organism to diverse pathologies viz
Cancer, Heart disease, Neurodegenerative disease
including Cataract (Thomas et al., 2000; Ernesto et
al., 1987) Altered calcium homeostasis due to
oxidative stress leads to increased intracellular
calcium levels and in turn leads to cataract formation
(Wride, 1996; Nakamura et al., 2000)
The aim of the study was to evaluate the total
antioxidant activity and calcium levels in diabetic
patients lens depending on type of cataract and to
correlate the levels of calcium with the antioxidant
activity in progression of cataract formation.
MATERIALS AND METHODS
The study included fifty diabetic subjects
having cataract, aged between 45 -70 years of either
sex. Later they were grouped into group 1- immature
diabetic cataract patients and group 2- mature
diabetic cataract patients based on ophthalmoscopic
examination by ophthalmologist. Patients with
57 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
Deepa et al. (2012)
history of steroid intake, other ophthalmic disease,
and systemic disorders were excluded from the study.
The study was approved by the Institutional Time
Bound Research Committee. A written informed
consent was taken from the subjects. Postoperatively
cataractous lens were collected in ice cold 0.9%
Normal saline container and transferred in ice box to
the place of analysis and analyzed on the same day
immediately for the following parameters: For
determining the antioxidant activity and calcium
levels in lens, the lens was homogenized with 1.2ml
ice cold 0.01M phosphate buffer pH 7.4. Mixture was
centrifuged at 2500rpm for 10mins. 100µl
supernatant was used for analysis. The total lens
antioxidant activity was determined by Koracevic’s
method (Koracevic et al., 2001) the assay measures
the capacity of antioxidant to inhibit the production of
thiobarbituric acid reactive substances from sodium
benzoate under the influence of the oxygen free
radicals derived from Fenton reaction. The reaction
was measured spectrophotometrically at 535nm.
Calcium in the lens was determined by
Cresolphthalein complexone method (Stern, 1957)
using calcium kits from Aspen Lab Pvt Ltd.
Cresolphthalein complexone a metal complex dye
reacts with calcium ions in alkaline medium forming a
purple color. Intensity of the color formed is directly
proportional to the calcium concentration and was
measured photometrically at 570nm. Since we
analyzed in lens tissue we need to express the levels
of total antioxidant activity and calcium in milligrams
of proteins .So protein content was determined by the
Lowry’s method (Lowry et al., 1951) blue color
obtained was measured spectrophotometrically using
Bovine albumin as standard
Statistical Analysis
The data was statistical analysis by epi-info
software using Analysis of Variance [ANOVA]. Since
two way ANOVA showed that the interaction between
cataract and maturity is significant, the comparison
between the type of cataract and maturity was done
separately using independent ‘t’ test. The results were
expressed as Mean ± Standard deviation.
The significance level at p<0.05 was
considered statistically significant. To correlate the
levels of calcium with the antioxidant activity,
Pearson’s correlation co-efficient was worked out.
Research Article Deepa et al. (2012)

Table 1. Comparison of parameters between immature and mature cataract.

N Immature N Mature
Dry weight of lens 24 56±1.79 26 70±2.57**
Total Proteins 57±0.56 65±0.66*
(mg/lens)
Antioxidant 1.320± 0.208 0.820± 0.085***
Activity
(mg/mg of protein)
Calcium 24 0.805± 0.416 26 1.024± 0.056***
(mg/mgof protein)
***=p<0.001

RESULTS number of studies have focused on the individual

The comparison of the antioxidant activity
and calcium levels between mature and immature
cataractous lens are presented in Table 1. A
significant reduction in the Antioxidant activity in
mature cataractous lens was observed with the mean
value of 0.82 mg/mg of proteins against the value of
1.32mg/mg of protein seen in immature lens. A
significant increase in the lens calcium was observed
in mature cataract. The mean value being
1.024mg/mg of protein against 0.805mg/mg of
protein observed in immature cataract. A negative
correlation was seen between lens antioxidant
activity and lens calcium and r value was -0.966.
DISCUSSION
Dysfunctional neuro-endocrine–endocrine
interactions contribute to the disturbances in trace
element metabolism and cause severe complications
in diabetes mellitus (Tallman et al., 1999). The
present results showed that the levels of calcium
were statistically increased in the mature cataractous
lens compared with immature diabetic cataract
patients (p<0.001), and the antioxidant activity is
statistically decreased in mature diabetic cataractous
lens (p<0.001).
Aksoy et al. (2001) have reported, a reduced
total antioxidant status in aqueous humor in patients
with diabetic cataract than those with senile cataract
(Aksoy et al., 2001). A much reduced antioxidant
activity in diabetic patients could be explained by
hyperglycemia induced complication such as reduced
gene expression of antioxidant enzymes and inactivity
of existing enzymes by glycation (King et al., 1996).
Thus resulting, overall reduction in antioxidant levels
which fails to defend oxidative stress, there by
accelerating the process of cataractogenesis. A
58 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
antioxidants. A decreased level of erythrocyte
glutathione peroxidase, glutathione reductase has
been reported in diabetic cataract. Likewise decrease
levels of SOD, Catalase, GSH; Glutathione peroxidase
has also been reported in lens (Nishikawa et al., 2000;
Chandrasena et al., 2006). The role of calcium in
lenticular metabolism has been studied by many
workers in animal models and their study results are
as follows:
P.G. Biju (Biju et al., 2007) and Lifei Wang
(Wang et al., 2001) studies showed that the increased
lenticular calcium is due to altered calcium ATPase
Pump activity due to oxidative stress, which in turn
leads to activation of calpain proteases which is
responsible for proteolysis. In Fein et al. studies,
increased lenticular calcium causes aggregation of
high molecular weight proteins (Fein et al., 1979).In
G-Duncan et al, KR Hightower et al, (Duncan et al.,
1993; Hightower et al., 1980) studies the altered
calcium homeostasis is due to altered calcium ATPase
and sodium calcium exchange mechanism and
thereby affecting calcium transport across lens.
Investigations by Tang (Tang et al., 2003) have shown
that the traces of calcium in lens are in some way
related to the maintaince of normal permeability and
regulation of dynamic equilibrium between ionic
constituents of its lens and its surrounding fluid.
Thus there is a significant increase in calcium
content with the onset of maturity is due significantly
decreased antioxidant levels. Altered calcium
homeostasis, due to oxidation of the regulatory
proteins or by damage to the plasma membrane by
lipid peroxidation, which thereby affects the
membrane functions and structure, may be the cause
for increase in calcium levels in these patients. In lens,
calcium is essential for various lens fiber cell
processes including its differentiation. An optimum
concentration is essential for activation of Ca-ATPase
pump. Altered calcium homeostasis due to oxidative
Research Article Deepa et al. (2012)

Figure 1. Comparison of lens antioxidant activity between Immature and Mature diabetic cataract patients

Figure 2. Comparison of lens calcium levels in Immature and Mature Diabetic Cataract patients

stress leads to increased intracellular calcium levels
and in turn leads to cataract formation by following
mechanisms, increased intracellular calcium interacts
with cytoplasmic proteins causing protein oxidation
and aggregation of high molecular weight proteins.
This leads to increase in soluble protein aggregates.
Elevated calcium levels leads to widespread
activation of proteases particularly calpain, leading to
proteolysis of crystallin. Increased calcium reduces
the calcium ATPase pump activity and sodium
potassium ATPase activity thereby altering the
membrane permeability of lens (Thomas et al., 2003)
The present data revealed that hyperglycemia
produced marked oxidant impact as evident by the
significant decrease in antioxidants status in lens.
This definitely proves that oxidants play an important
role as well in genesis of cataract. Hence, estimation
of total antioxidant activity can serve as good
predictive marker in assessing the disturbed redox
59 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
status which is responsible for opacification of lens
and also calcium plays an important role in causation
of diabetic cataract.
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